The gram-positive bacterium Staphylococcus epidermidis is the most common pathogen in hospital-acquired infections. The costs related to infections caused by Staphylococcus epidermidis in the hospital setting are enormous and represent a major health care burden. Most infections caused by S. epidermidis occur after the insertion of indwelling devices such as catheters or prosthetic heart valves. In these cases, the ability of S. epidermidis to form biofilms represents the most important virulence determinant. In a biofilm, the bacteria are dramatically less susceptible to antibiotic treatment and to attacks by human immune defenses. For these reasons, S. epidermidis infections are very difficult to eradicate. To provide the scientific basis for the development of drugs interfering with biofilm formation, we are investigating the molecular biology, biochemistry, and epidemiology of biofilm formation in S. epidermidis. This includes studying specific factors contributing to biofilm formation, their regulation, and the interaction of biofilm-forming S. epidermidis strains with the host: 1. Global approaches to understand S. epidermidis pathogenesis We analyzed the published S. epidermidis genome sequence, annotated the genome, and constructed a microarray comprising all putative genes with a length of > 60 bp. Gene expression profiling of cells in a biofilm demonstrated that a biofilm represents a status of generally reduced metabolism and increased expression of specific protective factors. Furthermore, to discover factors that determine invasiveness of S. epidermidis, we performed comparative genomic hybridization using strains isolated from S. epidermidis infection and compared to commensal strains. The biological roles of selected factors with a higher frequency among clinical strains will be investigated further. 2. Regulation of biofilm formation We focused our research on the role of the agr quorum-sensing system in biofilm formation. We could show that agr mutants show increased biofilm formation in S. epidermidis, demonstrating a negative influence of quorum-sensing on biofilm formation in this organism. As a consequence, an isogenic mutant of the agr quorum-sensing system showed increased colonization of indwelling medical devices in an animal infection model. Furthermore, agr mutants were found to occur naturally and significantly more frequently among strains isolated from infection of indwelling medical devices. Our results demonstrate a novel role for quorum-sensing control of biofilm formation in S. epidermidis. In a collaboration with Dr. Q. Gao?s group from Shanghai, China we investigate the function of another global regulatory system, luxS. We found that luxS influences biofilm formation in a way similar to agr, i.e. the absence of luxS leads to more pronounced biofilm formation. However, in contrast to agr, luxS impacts the formation of S. epidermidis biofilms via transcriptional control of the expression of the exopolysaccharide PIA. 3. PIA PIA is the main determinant responsible for intercellular adhesion, which is considered the second step in biofilm formation. We detected a novel role of PIA in immune evasion. We found that PIA protects against phagocytosis and killing by human PMNs and also protects against cationic and anionic antibacterial peptides. PIA is the first defined factor to be found in S. epidermidis that protects against major mechanisms of the human innate immune system. PIA is a homopolymer of partially de-acetylated N-acetyl-glucosamine units. The basis and biological function of deacetylation is unknown. We could demonstrate that a protein, IcaB, is localized on the S. epidermidis surface and is involved in PIA deacetylation, most likely representing the PIA deacetylase. Of note, deacetylation of PIA was crucial for biofilm formation, tissue colonization, immune evasion processes, and virulence in an animal infection model. IcaB is the first factor found that modifies bacterial exopolysaccharide to ensure pathogen success in biofilm-associated infection. 4. Role of a poly-gamma-glutamate capsule in S. epidermidis We demonstrated that S. epidermidis, as the only human pathogen other than Bacillus anthracis, produces a capsule-like structure of poly-gamma-glutamate (PGA) to shelter the bacteria from phagocytosis. Additionally, PGA protected S. epidermidis from antibacterial peptides of human skin and high salt concentration. PGA in S. epidermidis thus has a hitherto unique role in facilitating survival in both the natural habitat of the bacteria and during infection. In contrast to PIA, which is only produced by some S. epidermidis strains, PGA was synthesized by all tested strains of commensal and clinical origin, representing the first widespread mechanism of immune evasion discovered in S. epidermidis. Importantly, an animal model indicated a crucial role for PGA in S. epidermidis device-related infection, suggesting that PGA represents an excellent target for therapeutic maneuvers aimed at treating disease caused by S. epidermidis. 5. Phenol-soluble modulins (PSM): role in immune modulation and biofilm formation PSMs are small, amphipathic peptides with strong immuno-modulatory properties. We have expressed the individual peptide components of PSM and determined their pro-inflammatory capacities in collaboration with S. Klebanoff (University of Washington). We found that PSM production is wide-spread and that the relative proportion of the individual components is constant. We determined quorum-sensing control of the production of PSM and found an extremely tight regulation of PSM production by the agr quorum-sensing system, resulting in the complete absence of PSM and PSM-like peptides in agr mutants. We propose that quorum-sensing control of PSM production helps to provoke an optimal level of inflammation that contributes to bacterial survival. Additional results suggested an important role of the PSM peptides not only as immuno-modulatory compounds, but also in influencing biofilm structure, possibly in the insufficiently understood process of detachment of cell clusters from a biofilm, which is believed to be of major importance to the spread of an infection in vivo. We are currently investigating this effect using, among other methods, confocal laser scanning microscopy of biofilms. Finally, we have identified PSM-like molecules in the important human pathogen Staphylococcus aureus and are investigating the biological roles of these peptides.